Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain
Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain
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DOI：
10.3390/v12080840
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发表时间：
2020-07
期刊：
影响因子：
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通讯作者：
Eriko Ohsaki;K. Ueda
中科院分区：
文献类型：
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作者：
Eriko Ohsaki;K. Ueda
乙肝病毒聚合酶似乎很难得到充分的表达和纯化，这长期以来阻碍了基于该聚合酶性质的抗乙肝药物的产生。到目前为止，还没有开发出用于抗乙肝病毒聚合酶药物筛选的有用系统。在本研究中，我们成功地获得了高纯度的逆转录酶(RT)结构域，该结构域具有模板/引物和底物结合活性，并建立了利用纯化的RT蛋白寻找新的聚合酶抑制剂的高通量筛选(HTS)体系。为了检验检测系统是否提供了可靠的结果，我们使用具有药理活性的化合物进行了小规模筛选。结果，试点筛查确定了已知的抗病毒聚合酶制剂。然后，我们筛选了2万种化合物，并新确定了4种匹配的化合物。其中几个化合物不仅抑制HBVRT底物和/模板/引物结合活性，而且还抑制具有延伸活性的Moloney小鼠白血病病毒RT活性。最后，即使在基于细胞的测试中，这些候选药物也显示出了有效性。我们的筛选系统为寻找抗乙肝病毒的候选抑制剂提供了有用的工具。
Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.